Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium

Recent advances in transient transfection protocols using polyethylenimine (PEI) as a transfection reagent have led to the development of economical methods that provide yields sufficient for industrial production of proteins for many preclinical needs. There are many variables that can be optimized to improve protein expression in transient transfection, and one of the most critical is the medium in which the cells are grown. While transfection with PEI works well in media containing serum, the biopharmaceutical industry is moving away from animal-derived components in media. A number of serum-free media have been found to allow transient transfection, but many others do not for reasons that are not clear. Thus, knowledge of the components of serum-free media that can cause inhibition of PEI-mediated transient transfection would be useful for media development. In this study, an analysis was performed of various components of a serum-free medium used for Chinese hamster ovary cells in which PEI-mediated transient transfection was inhibited. We found that an iron supplement added to the medium was responsible for the inhibition. Further investigation showed that iron (III) citrate, a common iron chelator found in serum-free medium, was the specific component that caused the effect. Further, we showed that inhibition of transient transfection was caused by iron (III) citrate specifically, rather than citrate or iron alone. Finally, we showed that various iron chelators in serum-free media other than iron (III) citrate do not inhibit antibody expression.